): Cryptococcus neoformans is a ubiqutious fungal pathogen that causes life-threatening disease in man. Althogh AIDS patients and immunodeficient individuals are the most susceptible, normal individuals acquire this life-threatening disease as well. Without T lymphocyte function, currently available antifungal drugs do not cure the disease. Moreover, multiple drug resistance is appearing on clinical isolates. Even though several virulence factors have been identified, those factors do not account for all aspects fo the pathogenesis. C. neoformans is an encapsulated Basidiomycete, making it unique genetically among human pathogens. Consequently, gene databases for the other fungi do not serve as guides for studying C. neoformans. A thorough understandind of the genome of C. neoformans would be an extremely valuable resource for moving forward research on C. neoformans. The most efficient, econominal approach to generating a useful genetic inventory is through a combination of expressed seuqence tags (ESTs) and genomic sequencing. Although low density seuqencing of the C. neoformans genome is being done, the annotation with be completely dependent on reliable gene-fingidn software. Based on preliminarydata, it is hypothesized that C. neformans intron/exon junctions are sufficiently different from known eukaryotic seuqneces that gene-finding software will have to be trained for C. neoformans before the software will be useful. We proposed to: 1) generate ESTs from a primary JEV21 cDNA library, 2) prepare a JEC21 normalized library, 3) sequence 4000 cDNAs from the normalized library, 4) fully sequence by primer walking PCR products from selected cDNA clones and genomic DNA and identify the intron/exon boundaries, and 5) train gene-finding sofltware to recongize intron/exon junctions of C. neoformans. ESTs will be useful to identify ORFs, new drug targets, new virulence markers, cell surface adherence factors, and metabolic and signaling pathways.
